Recovery of mangrove polluted by Hydrocarbons

Recovery of mangrove polluted by Hydrocarbons

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Mangroves are subject to different risks of contamination, especially the petrochemical industry. Pollutants such as oil, heavy metals, industrial and urban waste, directly affect mangrove vegetation, altering the respiratory and osmoregulatory capacity of the roots, and can cause the death of plants.


Mangroves develop naturally, in the intertidal strips of the tropical and subtropical coasts of the planet, thus constituting a complex ecosystem due to the interaction of hundreds of species of all taxonomic levels, from microorganisms to mammalian species. large and showy, including among all these, hundreds of kinds of fish that are the livelihood of many communities. All the species that interact in the mangrove have their specific basin and determined functions, of vital importance for the dynamics and functioning of these ecosystems (FAO - INDERENA, 1984).

These regions are subject to different risks of contamination due mainly to industrial and port activity. Within these activities, the petrochemical industry is an important source of contamination for these areas (Carvalho 2002). Pollutants such as oil, heavy metals, industrial and urban waste, directly affect mangrove vegetation, altering the respiratory and osmoregulatory capacity of the roots, and can cause the death of plants. Among the results that can be obtained due to mangrove contamination are, among others, a decrease in water quality, productivity, biodiversity, landscape and aesthetic values ​​of marine ecosystems (Fildeman, 1999).

Mangroves are the ecosystems most sensitive to oil pollution, due to the toxicity of the spilled products and their ability to cover roots, pneumatophores, tree bark, etc., thus interfering in the metabolism of the plant, specifically in gaseous exchange. In the same way, it causes denaturation of cell membranes, reducing perspiration and photosynthesis, affecting respiration and reducing plant growth (LAFFER Encyclopedia, 2000).

In the Central Petroleum Drilling and Extraction Company, located on the north coast of the Republic of Cuba, a breakdown occurred in the old pipeline that transported crude oil to the base of super-tankers, producing a dumping of this product to an area of nearby mangrove, which caused severe soil contamination. Because mangroves are highly sensitive sites and in which extreme measures are applied for their conservation and protection, “in situ” sanitation technologies are required to avoid damage to the marine ecosystem.

One of the possible technologies to apply was the application of products with a high microbial load to promote the in situ biodegradation of hydrocarbons. Among these are the sewage sludge or digested sludge obtained in the treatment process of domestic and industrial wastewater.

The sludge is usually a liquid or semi-solid with a variable solid content, depending on the treatment operations received, and which is usually between 0.25% and 12% by weight (Gutiérrez, J. 2003).

Sludge (sludge) is formed mainly by the substances responsible for the unpleasantness of untreated wastewater; The fraction of sludge to evacuate, generated in the biological treatment, is mainly composed of the organic matter present in it, although in a different way from the original, only a small part of the sludge is composed of solid matter (Gutiérrez, J. 2003).

The value of the mud as a fertilizer is based mainly on the content of nitrogen, phosphorus and potassium, which must be determined in those cases in which the mud is to be used as a soil conditioner (Gutiérrez, J. 2003). Interest in the application of sludge to the ground has increased lately as a consequence of the lower availability and viability of other sludge management operations such as depositing in controlled landfills. Proper management of sludge is necessary due to the reduction in the availability of authorized or controlled landfills and the legal efforts to reuse its nutritional properties (Palacios, 2003).

In addition, by adding sludge from water treatment plants to the soil, the content of organic material increases, since it can generate a reduction in surface water runoff, favoring moisture retention, thus improving biological activity and therefore mineralization processes. of nutrients. Microbial activity favors structural stability, since microbial tissue can form a network around groups of mineral particles. (Dibble and Bartha, 1979).

The residual mud improves other properties of the soil such as: pH, real density, apparent density, lower limit of plasticity, plasticity index and electrical conductivity.

The general objective of this work is the recovery of the mangrove soil contaminated by hydrocarbons by applying the bioremediation process through the bioaugmentation technique, using as a bio-product the residual sludge or digested sludge obtained in the treatment of sewage sewage Taíno 1, belonging to the company Aguas Varadero.

The specific objectives of this work are:

  1. Use the wastewater from the process of obtaining henequen fibers as a surfactant. Determination of surface tension to residual water from henequen production.
  2. Isolation and identification of strains of degrading microorganisms from the hydrocarbon fractions in the residual sludge.
  3. Carry out the analytical, chemical and microbiological monitoring of the bioremediation treatment applied to the mangrove soil contaminated by hydrocarbons.

Materials and methods.

1- Treatment of mangroves impacted by hydrocarbons

The mangrove swamp contaminated by heavy hydrocarbons covers an area of ​​500 m2 of an oil drilling and extraction company located in the north of the Republic of Cuba.

3 inoculations of residual sludge or digested sludge obtained in the sewage treatment plant were carried out in the months of January, February and March with 10, 6 and 8 m3, respectively, in order to maintain the levels of the sludge in soil. The sludge was transported in a 25 m3 capacity tank car and was applied by using hoses coupled to the tank cars as shown in Fig. 1.

Figure 1. Sludge inoculation

To increase the bioavailability of the hydrocarbon present in the mangrove soil to the action of microorganisms, wastewater from the production of henequen fibers was used, the surface tension of which was determined in a tensiometer from the North American firm, CENCO , model 70535. The wastewater was previously neutralized with calcium hydroxide to neutral pH. Wastewater was added manually, and at a rate of 0.12 l of wastewater per m2 of impacted mangrove soil, on three occasions.

2- Sample taking

The soil sample was taken using a star type sampling recommended by the Japanese specialist Dr. Itaru Okuda (OKUDA, 2002). The samples were collected and packed in aluminum bags (ISO 5667,1994) and preserved in freezing until their subsequent processing and analysis. Furthermore, they were homogenized, dried and sieved.

The sample of residual sludge or digested sludge was taken promptly in the final discharge of residual sludge or digested sludge to the drying bed of the urban wastewater treatment plant. The sample was packed in sterile 1 liter bottles and kept cold until its use in the microbiology laboratory (ISO 10381–6, 1993). Subsequently, the microorganism count was carried out, and the concentration of nutrients was determined.

3- Analytical monitoring of treatment

To determine the effectiveness of the applied treatment, the following physical-chemical analyzes were carried out:

The% removal of hydrocarbons was calculated according to the following expression:

Initial H / C Concentration - Final H / C Concentration

% Removal = ———————————————————————- * 100

Initial H / C Concentration

4- Reforestation of the recovered mangrove area

Mangrove seeds (pods) were sown manually (Figure 2) to reforest the recovered mangrove zone. They were selected according to the recommendations of people who live in the region.

Figure 2 Mangrove seeds

5- Isolation of bacteria strains from digested sludge or residual sludge

The most representative colonies grown on petroleum agar were seeded by depletion in tryptone soy agar medium and incubated at 37 o C for 24 h. For the cultural characterization, the colonies were observed with a stereoscope and to define the morphological staining characteristics, the Gram stain was performed. (Bergey's Manual, 1974)

  • Characterization of degrading strains

Degradation of Resins and Asphaltenes: To determine the capacity of these strains to degrade heavy crude oil, 1% of distillation residue greater than 347 oC and distillation residue greater than 465 oC of Crudo Seboruco were added in tubes with 9 ml of Solanas medium. The medium was inoculated with a culture roast of the strain, the tubes were incubated for 21 days at 37 ° C with a growth indicator.

Aliphatic hydrocarbons: For the determination of the capacity to use aliphatic petroleum hydrocarbon fractions as the sole carbon source, 1% kerosene and 1% Isooctane were added separately in tubes with 9 ml of Solana medium. The medium was inoculated with a culture roast of the strain and incubated for 21 days at 37 ° C with growth indicator.

Aromatic Hydrocarbons: For the determination of the utilization capacity of aromatic hydrocarbon fractions, 1% Naphthalene (polyaromatic) and 1% Toluene (Aromatic alkylated) were used separately in Solanas basal medium. Naphthalene, as it is a solid insoluble in water, was first dissolved in acetone and then added to the medium. The medium was inoculated with a culture roast of the strain and incubated for 21 days at 37 ° C with growth indicator.

Identification of degrading strains: Identification kits (API 20 NE, 2002) were inoculated from fresh cultures of the strains that grew in all the characterization media for the identification of Gram-negative non-enterobacterial microorganisms.

Results and Discussion.

Waste water from henequen fiber production

Table 1 reports the results of the surface tension test carried out on the wastewater from the production of henequen fibers.

Table 1. Surface Tension Values

In the production of henequen fibers, wastewater is obtained that can very well be used as surfactants due to the presence of saponins, these wastewater has a very low pH (<4) and infiltrates the water table without receiving any treatment. . The results obtained from the surface tension test indicate that once the pH is adjusted to neutral values, these wastewater can be used as surfactants because the surface tension value of wastewater is lower than that of distilled water, as is as seen in the table above.

Waste sludge from the Taíno 1 domestic sewage treatment plant

Table 2 shows the microbiological characterization of the residual sludge from the Taíno 1 domestic sewage treatment plant. The high hydrocarbon degrading microbial load present in this waste can be observed, on the order of 107, making it possible to use it as inoculum in the treatment of soils contaminated by hydrocarbons.

Table 2. Microbiological characterization of the digested sludge, expressed in CFU / g.

Monitoring of the Bioremediation treatment applied to the mangrove soil contaminated by oil.

Table 3 reports the physical-chemical and microbiological results of the analytical monitoring carried out on the mangrove soil contaminated by hydrocarbons.

Table 3. Analytical monitoring of contaminated mangrove soil.

HCTP: Total Petroleum Hydrocarbons.

Figure 3. Impacted mangrove

A marked decrease in the concentration of total hydrocarbons is observed over time due to the combined application of the mud with chemical surfactants, residual water from the production of henequen fibers, which favored a better bioavailability of the hydrocarbon to the attack of the microorganisms present in the residual sludge. At the end of the period, 359 days, it was possible to reduce the concentration of total oil hydrocarbons below 1%, a value that a soil remediated by the bioremediation process in the state of Louisiana, USA must meet. It should be noted that in the mangrove soil control sample, where the residual sludge was not applied, the variation in the hydrocarbon content is almost zero, which shows the biodegradative properties of the applied product.

In addition, the presence of high concentrations of total microorganisms and hydrocarbon biodegraders in the treatment time, a factor that enhances decontamination in soils impacted by fuels.

Figure 4. Treated mangrove

Figure 4 qualitatively shows the mangrove soil recovered by applying the bioremediation process using the bioaugmentation method compared to the mangrove impacted at the beginning of the treatment (Fig. 3).

High by hundreds of reduction, 76.78%, of the heaviest fractions of oil, Resins and Asphaltenes, these compounds more resistant to the degradative process, were achieved, in comparison to results obtained in the Control sample.

Isolation of bacteria strains from digested sludge or residual sludge

Table 4 and 5 show the result of the physical-chemical and microbiological characterization of the digested sludge, total count of the colonies of aerobic bacteria, fungi and yeasts and degrading microorganisms expressed in CFU / g.

Table. 4. Microbiological characterization of the digested sludge, expressed in CFU / g.

The number of aerobic bacteria reached values ​​of 109 CFU / g, fungi and yeasts up to 105 CFU / g and hydrocarbon degrading microorganisms were high with values ​​reaching up to 106 CFU / g. These results correspond to the wide flora characteristic of the soils (Bergeys, 1994).

Table. 5. Physical - chemical characterization of the digested sludge

HCTP: total petroleum hydrocarbons

This substrate constitutes a rich source of organic matter with high content of nitrogen, phosphorus and potassium where a high percentage of fats, oils and total hydrocarbons dissolved in the sediment appear with high bioavailability and a suitable pH for the development of an abundant adapted microbiota. to the consumption of crude oil and its derivatives.

Eleven apparently different strains were isolated, in table 6 and the morphological cultural characteristics of each one of them appear.

Table 6. Cultural and staining characteristics of the isolated strains

As can be seen in table 6, the bacteria presented diverse colonial morphology, which is in accordance with the nutritional richness of the river mud from which they come.

The microscopic study showed bacillary, coccobacilli, Gram positive and negative forms, smooth and irregular margins, convex and flat, of green, yellow and pink colors. The isolated bacteria turned out to be 5 positive coccobacilli, 3 negative coccobacilli. A positive bacillus and 2 negative bacilli also appeared.

  • Characterization of the isolated hydrocarbon degrading strains

Strains LR-1, LR -2, LR -3, LR -5, LR -7c, LR -9 and LR -13 grew in all media with the different substrates as the sole source of carbon and energy (Table 7), These bacteria had an optimal growth at the end of the incubation with turbidity and color change (Fig. 13), being able to degrade all the hydrocarbon fractions to which they were exposed, therefore they constitute promising strains to be used for different degradative purposes. .

This nutritional versatility is very important since few microbial species reported with the potential to degrade various types of hydrocarbons since most hydrocarbon degrading strains can mineralize polyaromatic or aliphatic compounds but not both, which suggests that these two pathways they can be exclusive in many cases. However, genes coding for the enzymes alkane-monooxygenase and catechol 2,3 dioxygenase have been observed in many bacteria, indicating that degradative pathways for alkanes and alkylated simple aromatic hydrocarbons can be found simultaneously (Whyte, Bourbonnié and Creer, 1997).

Table 7. Results of the characterization of the strains isolated from the residue with the different hydrocarbons

Reforestation of the recovered mangrove area

When evaluating the sowing of the yellow mangrove seeds planted in the recovered mangrove area, it is observed that, at the time of writing this report, they are in a state of germination, with green coloration, indicating that the seeds are in process. growth and development, so it is recommended to periodically evaluate the reforestation of the recovered area. In addition, it is necessary to comprehensively evaluate the flora and fauna of the mangrove in order to know whether the application of residual mud negatively affected the life of animals and plants native to these ecosystems.


  1. The bioremediation by the bioaugmentation technique applying as a bioproduct a residual sludge from a domestic wastewater treatment plant, to a mangrove area impacted by hydrocarbons, was satisfactory as total oil hydrocarbon values ​​of less than 1% were achieved.
  2. The digested sludge from wastewater treatment plants constitutes an important source of hydrocarbon degrading microorganisms, for which its use in the bioremediation process of soils impacted by hydrocarbons is promising.
  3. Significantly high% hydrocarbon removal was achieved for the applied bioremediation process.
  4. 5 Gram negative strains were identified, resulting in genera commonly isolated from soil and mud.
  5. Of all the bacteria isolated from the residual sludge studied, seven were the ones with the best biodegradation results using crude oil and its derivatives as the sole source of carbon and energy. The strains called LR-1, LR -2, LR -3, LR -5, LR -7c, LR -9 and LR -13, are promising for their application in different Bioremediation processes.


  • Evaluate the flora and fauna of the recovered mangrove area by applying digested sludge or sludge from domestic and industrial wastewater treatment plants as inoculum or bioproduct.
  • Toxicological evaluation of the recovered mangrove soil.

MSc. José Alfonso Álvarez González, Technician Gisela Novoa Rodríguez, Engineer Roberto Romero Silva, Dr. Miguel A. Díaz Díaz, Lic. Sandra Millar Palmer, Tec. Ahiram López Díaz, Tec. Cristina Laffita Rivera.


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Video: Australias Sustainable Ocean Economy webinar (June 2022).


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